Expression, purification and applications of staphylococcal Protein A fused to cellulose-binding domain

Because staphylococcal Protein A (ProtA) binds specifically to IgG, it has been used for many immunological manipulations, most notably antibody purif ication and diagnostics. Immobilization is required for most of these appli cations. Here we describe a genetic-engineering approach to immobilizing Pr otA on cellulose, by fusing it to cellulose-binding domain (CBD) derived fr om the cellulose-binding Protein A of Clostridium cellulovorans, The bifunc tional fusion protein was expressed in Escherichia coli, recovered on a cel lulose column and purified by elution at alkaline pH. ProtA-CBD was used to purify IgG from rabbit serum and its ability to bind IgG from different so urces was determined. The bifunctional chimaeric protein can bind up to 23. 4 mg/ml human IgG at a ratio of I mol of ProtA-CBD/2 mol of human IgG, and can purify up to 11.6 mg/ml rabbit IgG from a serum. The ability to bind fu nctionally active CBD-affinity reagents to cellulosic microtitre plates was demonstrated. Our results indicate that a combination of CBD-affinity reag ents and cellulosic microtitre plates is an attractive diagnostics matrix f or the following reasons: (i) cellulose exhibits very low non-specific bind ing; and (ii) CBD-fusion proteins bind directly to cellulose at high densit y. A unique signal-amplification method was developed based on the ability of ProtA-CBD to link stained cellulose particles to primary antibody in a W estern blot.